A novel monocyte differentiation pattern in pristane-induced lupus with diffuse alveolar hemorrhage

Pristane causes chronic peritoneal inflammation resulting in lupus, which in C57BL/6 mice is complicated by lung microvascular injury and diffuse alveolar hemorrhage (DAH). Mineral oil (MO) also causes inflammation, but not lupus or DAH. Since monocyte depletion prevents DAH, we examined the role of monocytes in the disease. Impaired bone marrow (BM) monocyte egress in Ccr2−/− mice abolished DAH, confirming the importance of monocyte recruitment to the lung. Circulating Ly6Chi monocytes from pristane-treated mice exhibited increased annexin-V staining in comparison with MO-treated controls without evidence of apoptosis, suggesting that pristane alters the distribution of phosphatidylserine in the plasma membrane before or shortly after monocyte egress from the BM. Plasma membrane asymmetry also was impaired in Nr4a1-regulated Ly6Clo/− ‘patrolling’ monocytes, which are derived from Ly6Chi precursors. Patrolling Ly6Clo/− monocytes normally promote endothelial repair, but their phenotype was altered in pristane-treated mice. In contrast to MO-treated controls, Nr4a1-regulated Ly6Clo/− monocytes from pristane-treated mice were CD138+, expressed more TremL4, a protein that amplifies TLR7 signaling, and exuberantly produced TNFα in response to TLR7 stimulation. TremL4 expression on these novel CD138+ monocytes was regulated by Nr4a1. Thus, monocyte CD138, high TremL4 expression, and annexin-V staining may define an activated/inflammatory subtype of patrolling monocytes associated with DAH susceptibility. By altering monocyte development, pristane exposure may generate activated Ly6Chi and Ly6Clo/− monocytes, contributing to lung microvascular endothelial injury and DAH susceptibility.

Differentiation of Ly6C − monocytes is driven by CCAAT-enhancer binding protein β (C/EBPβ) via activation of the transcription factor nuclear receptor subfamily 4 group A member 1 (Nr4a1) (Hanna et al., 2011;Mildner et al., 2017). Along with Nr4a1, Ly6C − monocyte development requires Notch signaling (Hanna et al., 2011;Gamrekelashvili et al., 2016). In contrast to the role of Ly6C hi monocytes as mediators of inflammation, Nr4a1-dependent Ly6C − monocytes patrol the vascular endothelium and promote the TLR7-dependent removal of damaged cells (Auffray et al., 2007;Carlin et al., 2013). Although they typically remain within blood vessels, they also can migrate across the endothelium into inflamed tissues (Auffray et al., 2007;Schyns et al., 2019). Ly6C − monocytes may represent a form of intravascular macrophage (Mϕ) (Ginhoux and Jung, 2014), as suggested by the expression of genes typical of tissue-resident Mϕ such as Apoe and Cd36 (Mildner et al., 2017). Although only a single population of Ly6C − monocytes is seen in the blood of unmanipulated B6 mice, less is known about Ly6C − monocytes in inflammatory settings.
Pristane and mineral oil (MO) both cause sterile peritoneal inflammation and an influx of Ly6C hi monocytes, which become Ly6C hi inflammatory peritoneal Mϕ . In pristane-but not MO-treated mice, the chronic inflammatory response evolves into lupus after several months . In pristane-treated B6 mice, but not BALB/c or other strains, the onset of lupus is complicated by lung microvascular injury and diffuse alveolar hemorrhage (DAH) H Zhuang, In Revision).
In contrast to the striking predominance of Ly6C hi Mϕ in the peritoneum of pristane-treated mice, Ly6C lo Mϕ predominate in MO-treated mice . A subset of the Ly6C − peritoneal Mϕ expresses CD138 (syndecan-1) . Ly6C lo CD138 + Mϕ from MO-treated mice have an anti-inflammatory phenotype and are highly phagocytic for dead cells. The significance of CD138 expression by Mϕ is unclear and it is not known whether CD138 is expressed by monocytes. We examined the distribution of CD138 expression in myeloid cells from the blood and inflamed peritoneum of pristane-and MO-treated mice.

Results
Although i.p. pristane injection induces lupus autoantibodies and renal disease in both B6 and BALB/c mice, only B6 mice develop DAH   , suggesting that it is mediated at least partly by monocytes and/or Mϕ. There is little information about monocytes in pristane-treated mice and their relationship to disease pathogenesis. Pristane-induced Ly6C hi monocyte recruitment from the BM to the peritoneum is Ccr2 dependent . A population of highly phagocytic peritoneal Ly6C − CD138 + Mϕ with an anti-inflammatory phenotype is more abundant in MO-vs. pristane-treated mice, whereas Ly6C hi CD138 − Mϕ are more abundant in pristane-treated mice . We examined the origin of these peritoneal Ly6C − CD138 + Mϕ.   . Ly6C hi monocytes also give rise to Ly6C −/lo monocytes in the circulation (Sunderkötter et al., 2004;Mildner et al., 2017), raising the question of whether peritoneal Ly6C −/lo CD138 + Mϕ are derived from circulating Ly6C −/lo monocytes or from peritoneal Ly6C hi Mϕ. CD138 + CD11b + cells were found in the blood of both pristaneand MO-treated B6 mice at 14 days, but were considerably more abundant in pristane-treated mice ( Figure 1A). In contrast, peritoneal CD138 + CD11b + Mϕ were more abundant in MO-treated mice than pristane-treated mice. BALB/c mice and B6 mice lacking B cells (μMT) do not develop pristane-induced DAH Barker et al., 2011). Like MO-treated B6 mice, both B6 μMT and BALB/c mice generally had fewer circulating CD138 + CD11b + cells and more peritoneal CD138 + CD11b + Mϕ than wild-type B6 mice ( Figure 1B). These observations suggested that circulating CD138 + CD11b + myeloid cells were recruited to the peritoneum and developed into CD138 + CD11b + Mϕ or alternatively, that CD138 + CD11b + Mϕ were generated locally in the peritoneum. As an initial step to distinguish between these possibilities, we carried out more extensive phenotyping of the circulating and peritoneal populations. Flow cytometry revealed three subsets of CD11b + Ly6G − cells from pristane-treated B6 mice in both the blood and the peritoneum (Figure 2A): CD11b + Ly6C hi CD138 − (R1); CD11b + Ly6C −/lo CD138 − (R2); and CD11b + Ly6C −/lo CD138 + (R3). Ly6C staining was higher on the peritoneal R2/R3 cells (Ly6C lo ) vs. blood R2/R3 (Ly6C − ) cells. In the blood of pristane-treated mice, all three subsets were CD43 + and TremL4 + , consistent with a monocyte phenotype ( Figure 2B,C). The blood R3 and R2 subsets expressed higher levels of CD43 and TremL4 than R1; R3 expressed higher levels than R2. In the blood CD115 was expressed at low levels, mainly on R3 cells ( Figure 2D). Interestingly, CD43, TremL4, and CD115 were highly expressed by the peritoneal R3, and to a lesser extent R2, subsets from MO-treated, but not pristane-treated, mice ( Figure 2B-D). As expected, surface staining for CD62L, which is expressed by Ly6C hi monocytes that have recently migrated out of the BM and not on Ly6C lo monocytes (Jakubzick et al., 2017), was stronger in R1 vs. R3 or R2 ( Figure 2E). Expression was highest in the circulating R1 subset. As classical monocytes are CD115 + Ly6C + CD43 lo whereas nonclassical monocytes are CD115 + Ly6C − CD43 hi TremL4 + (Jakubzick et al., 2017), we concluded that the circulating R1 cells were classical (Ly6C hi ) monocytes and the circulating R2 and R3 cells were subsets of non-classical monocytes. Circulating R3 cells also were CX3CR1 + CCR2 − ( Figure 2F,G).
Murine non-classical monocytes differentiate in the circulation from Ly6C hi precursors, have a Ly6C − CD43 + CX3CR1 hi phenotype and may represent terminally differentiated blood-resident Mϕ rather than true monocytes (Ginhoux and Jung, 2014). Consistent with that possibility, circulating R3 monocytes from pristane-treated mice expressed higher levels of the Mϕ marker F4/80 than R1 or R2 monocytes ( Figure 2H). However, another Mϕ marker (CD64, FcγR1a) was expressed at lower levels on R3 (and R2) vs. R1 ( Figure 2I). Peritoneal R3 Mϕ stained more intensely for F4/80, CD64, CCR2, and CX3CR1 than circulating R3 monocytes, and with the exception of CX3CR1, exhibited stronger staining than peritoneal R2 Mϕ. Together the data suggest that blood of pristane-treated mice contains classical monocytes along with two subtypes of non-classical monocytes distinguished by the presence (R3) or absence (R2) of CD138 staining. Peritoneal Mϕ also consisted of R1, R2, and R3 populations. Circulating R3 cells had a phenotype compatible with that of 'patrolling' monocytes but also had higher F4/80 staining than circulating R1 or R2 cells, consistent with early Mϕ differentiation. To further characterize the origin of this unusual monocyte subset, we examined CD138 monocyte development in Ccr2−/− and Nr4a1−/− mice.
As expected, circulating R1 monocytes were greatly reduced (but not absent) in pristane-treated Ccr2−/− mice ( Figure 3B). R3 monocytes also were greatly reduced in Ccr2−/− mice, suggesting that, like R1 monocytes, their migration from the BM to the circulation was Ccr2 dependent. In contrast to R1 and R3, the percentages of circulating R2 cells were similar in Ccr2−/− vs. wild-type mice. In PECs, R1 and R3 cells were absent and R2 cells were present at lower levels in Ccr2−/− mice vs. wildtype ( Figure 3B). Thus, the egress of R1 (Ly6C hi ) monocytes from the BM and the appearance of R3 (Ly6C − CD138 + ) monocytes in the circulation was Ccr2 dependent, whereas the circulating R2 monocyte subset (Ly6C − CD138 − ) was unaffected by the absence of Ccr2. R1 and R3 monocytes appeared in the circulation with different kinetics and all three peritoneal Mϕ subsets were reduced in Ccr2−/− mice, consistent with the possibility that they were derived from CCR2 + R1 monocytes.

Pristane-induced DAH is unaffected by the absence of Nr4a1
Induction of DAH by pristane requires monocytes and/or Mϕ . Pristane-treated Nr4a1−/− mice developed DAH at a frequency comparable to wild-type controls ( Figure 4E), suggesting that circulating CD138 + (R3) monocytes were not essential for the induction of DAH or were only needed in small numbers. In contrast, DAH was abolished in Ccr2−/− mice suggesting that, as in the inflamed peritoneum, migration of Ly6C hi monocytes from the BM to the lung is involved in the pathogenesis of DAH.
Further characterization of circulating monocytes from pristane-treated mice revealed that CD62L was expressed at high levels (as expected) on R1 and R4 cells ( Figure 6A). CD62L was absent on R3A/R3B and R6A/R6B cells, and was expressed at intermediate levels on R2A/R2B and R5A/R5B ( Figure 6A). Nr4a1, a transcription factor critical for generating non-classical monocytes (Hanna et al., 2011), was expressed intracellularly at high levels in R3A/R3B (and R6A/R6B), low levels in R1 (and R4), and intermediate levels in R2A/R2B (and R5B) ( Figure 6B). However, levels of Nr4a1 were low in R5A. Ly6C − LFA1 hi CCR2 lo CX3CR1 hi patrolling monocytes adhere and crawl along the endothelial cells in postcapillary venules, a process that is LFA-1 dependent (Auffray et al., 2007). Cx3Cl1 (fractalkine), the ligand for Cx3Cr1, is expressed on endothelial cells and mediates adhesion of monocytes to endothelial cells, whereas blocking antibodies to LFA-1 can detach crawling monocytes in Cx3cr1+/− mice (Auffray et al., 2007). Consistent with the possibility that they are patrolling monocytes, Cx3Cr1 and LFA-1 were expressed more highly by R3A/R3B (and R6A/R6B) monocytes than by the other subsets ( Figure 6C,D). Expression of CD11c, a differentiation marker expressed by dendritic cells as well as circulating 'foamy monocytes' containing intracellular lipid droplets (Wu et al., 2009;Xu et al., 2015), was greatly increased on the R3B (R6B) subset ( Figure 6E). Phenotypes of the R1, R2A/R2B, and R3A/ R3B subsets are summarized in Table 1. TLR7 ligand-stimulated TNFα production by Ly6C − CD138 + monocytes Phenotypic analysis suggested that the Ly6C − CD138 + subset may be a variant of patrolling monocytes, which encounter damaged endothelial cells respond to danger signals via TLR7 and then migrate into tissues where they undergo cMaf/MafB-regulated Mϕ differentiation and produce TNFα (Auffray et al., 2007;Ginhoux and Jung, 2014). Ly6C − cells are thought to promote focal endothelial necrosis followed by repair. We hypothesized that Ly6C − CD138 + monocytes might respond more aggressively to endothelial injury, potentially exacerbating damage rather than promoting resolution/repair. Along with their acquisition of the dendritic cell marker CD11c ( Figure 6E), the R3A/R3B (and R6A/R6B) subsets from pristane-treated B6 mice expressed considerably higher levels of TLR7 than other circulating monocyte subsets ( Figure 7A). These cells also expressed high levels of Treml4 ( Figure 7B), a protein that amplifies TLR7 signaling (Ramirez-Ortiz et al., 2015). As shown in Figure 7C, TLR7 ligand (R848)-stimulated intracellular TNFα staining was comparable in R3 monocytes vs. 'inflammatory' Ly6C hi monocytes. Moreover, TNFα staining was significantly higher in Ly6C − CD138 + (R3) monocytes vs. Ly6C − CD138 − (R2) monocytes ( Figure 7C, right). large peritoneal macrophages (LPMs) as a % of total peritoneal cells. Right, CD11b + Tim4 − CD138 + small peritoneal macrophages (SPMs) as a % of total peritoneal cells. (E) Frequency of diffuse alveolar hemorrhage (day 14) in pristane-treated WT, Nr4a1−/−, and Ccr2−/− mice. ****p < 0.0001 (Student's t-test). ns, not significant.  To investigate whether monocytes from pristane-treated mice were more prone to undergo apoptosis in response to TLR7 signaling, blood from 14-day pristane-or MO-treated mice was incubated for 20 hr with R848 (1 μg/ml) or PBS and then stained with anti-CD11b, Ly6C, and Ly6G antibodies plus PE-conjugated annexin-V. As shown in Figure 7D (left), the MFI of annexin-V staining was higher on Ly6C hi and Ly6C lo/− monocytes from pristane-vs. MO-treated mice. The MFI of neutrophils from pristane-vs. MO-treated mice was similar. Addition of R848 to the cultures did not significantly change the intensity of annexin-V staining. Histograms revealed that monocyte annexin-V staining was uniformly shifted to the right in pristane-vs. MO-treated mice, but there was only a single peak. In contrast, in neutrophils a second peak with more intense staining, most likely representing apoptotic cells, was seen, more prominently in pristane-vs. MO-treated mice ( Figure 7D, right, arrows). Monocytes did not exhibit this second peak. Annexin-V staining also was shifted to the right in freshly isolated monocytes (without in vitro culture) from pristane-vs. MO-treated mice (not shown). Intracellular staining of freshly isolated cells with anti-activated caspase-3 antibodies was negative (not shown), suggesting that the annexin-V + monocytes from pristane-treated mice were not apoptotic.
To further examine Nr4a1 regulation of Treml4 expression, RAW264.7 cells were cultured with LPS in the presence of Nr4a1 or control siRNA. As expected, Nr4a1 siRNA downregulated LPS-stimulated Nr4a1 mRNA expression at 1 hr ( Figure 10E). Nr4a1 siRNA also downregulated LPS-stimulated Treml4 mRNA expression at 3 hr. Adherent peritoneal Mϕ from untreated mice behaved similarly. As expected, Nr4a1 expression was minimal in PBS-treated adherent peritoneal Mϕ from Nr4a1−/− mice compared with wild-type controls ( Figure 10F, left). Unexpectedly, in adherent peritoneal cells of Nr4a1−/− mice, Nr4a1 increased slightly after LPS stimulation, probably due to the expression of a truncated fragment of the Nr4a1 gene encoding part of the N-terminal domain in B6.129S2-Nr4a1 t-m1Jmi /J (Nr4a1−/−) mice (Koenis et al., 2018). TremL4 expression was lower in PBS-treated adherent peritoneal cells from Nr4a1−/− mice vs. wild-type controls ( Figure 10F, right). Nr4a1 as well as Treml4 expression was significantly higher in both PBS-and LPS-treated adherent cells from wild-type mice vs. Nr4a1−/− mice ( Figure 10F).

Discussion
Transcriptional profiling of circulating monocytes from uninflamed B6 mice shows a single population of Nr4a1-regulated Ly6C − monocytes (Mildner et al., 2017). These cells mostly remain within the blood vessels where they patrol the vascular endothelium and promote TLR7-dependent repair of damaged endothelial cells (Auffray et al., 2007;Carlin et al., 2013). Previous studies have not fully addressed whether inflammation alters Ly6C − monocytes. We show that Ly6C − monocytes are more heterogeneous during inflammation than in the resting state. Pristane-induced lupus with DAH and small vessel vasculitis is a monocyte/Mϕ-dependent disorder involving lung microvascular endothelial injury H Zhuang et al., In Revision). Like pristane, MO causes a chronic inflammatory response, but it is not associated with DAH, pulmonary vasculitis, or endothelial damage. We found that Nr4a1-regulated patrolling (Ly6C − ) monocytes had different phenotypes in mice treated with pristane (CD138 + ) vs. MO (CD138 − ). Pristane directly affected cells of the monocyte/Mϕ lineage, increasing the expression of TremL4, a protein that amplifies TLR7 signaling and altering the normal plasma membrane asymmetry, as shown by increased annexin-V binding to phosphatidylserine on the plasma membrane's outer leaflet. Ly6C − monocytes from pristane-treated mice produced large amounts of TNFα in response to TLR7 ligand, but despite their high annexin-V staining, did not appear to be more susceptible to apoptosis than Ly6C − monocytes from MO-treated mice. The data suggest that pristane alters the activation state of Ly6C − patrolling monocytes, potentially influencing whether endothelial damage persists or is repaired.

Monocytes in mice with pristane-induced DAH
Pristane-induced lupus in B6 mice is complicated by severe lung disease closely resembling SLEassociated DAH with pulmonary vasculitis in humans Zamora et al., 1997). DAH and ANCA-negative small vessel vasculitis are abolished by depleting monocytes/Mϕ with clodronate liposomes and are absent in mice lacking immunoglobulin or C3, suggesting the involvement of immune complexes . DAH in SLE patients also shows evidence of an immune complex pathogenesis (Hughson et al., 2001;Al-Adhoubi and Bystrom, 2020). DAH in pristaneinduced lupus is associated with lung microvascular injury (Zhuang et al., 2017, in revision) and monocyte recruitment (Lee et al., 2019). We confirmed the importance of Ccr2 in DAH ( Figure 4E), underscoring the central role of Ly6C hi monocyte recruitment (Lee et al., 2019). R2 monocytes were unaffected by the absence of Ccr2, suggesting that they were dispensable for the induction of DAH ( Figure 3B and Figure 11). In contrast, R1 monocytes (deficient but not absent in Ccr2−/− mice) appeared to be required. R3 monocytes also were deficient in Ccr2−/− mice ( Figure 3B), but are derived from Ly6C hi precursors (Sunderkötter et al., 2004;Mildner et al., 2017). Nr4a1−/− mice had normal numbers of R1 monocytes and were susceptible to pristane-induced DAH ( Figure 4C, E and Figure 5A). R3 monocytes were greatly reduced (but not absent) in Nr4a1−/− mice ( Figure 4C), suggesting that they are not involved in DAH or are needed in only small numbers ( Figure 11).
Interestingly, circulating R1 monocytes were numerous in MO-treated B6 mice and pristane-treated B6 µMT and BALB/c mice, but they do not develop DAH ( Figure 11D). Thus, R1 monocytes were not sufficient to induce DAH. One explanation is that the R1 subset might differ in pristane-vs. MO-treated mice, as suggested by the altered annexin-V staining ( Figure 7D). Alternatively, the Nr4a1-regulated Ly6C − monocytes might differ. Consistent with previous studies in untreated mice (Mildner et al., 2017), Nr4a1-dependent Ly6C − CD11b hi (R6) cells in MO-treated mice were CD138 − and are likely to be patrolling monocytes monitoring the microvascular endothelium for damage (Auffray et al., 2007;Carlin et al., 2013). Thus, CD138 expression may be a marker for Ly6C − monocytes that have lost the ability to promote vascular repair. Consistent with this hypothesis, Ly6C − CD138 + monocytes express higher levels of Tlr7 and Treml4 than Ly6C − CD138 − monocytes and produce large amounts of TNFα in response to R848 ( Figure 7A-C). Annexin-V staining provided further evidence that circulating monocytes are different in pristane-vs. MO-treated mice ( Figure 7D). Annexin-V staining was substantially higher on Ly6C hi as well as Ly6C lo/− monocytes from pristane-vs. MO-treated mice, suggesting that plasma membrane asymmetry is altered by pristane treatment at an early stage (prior to the differentiation of Ly6C hi precursors into Ly6C − monocytes), with increased phosphatidylserine (PtdSer) exposure in the outer leaflet. Annexin-V staining of monocytes from pristane-treated mice was uniformly shifted to the right ( Figure 7D). In contrast, neutrophils from the same mice exhibited two populations of cells, one annexin-V lo and the other annexin-V hi . The annexin-V hi population was more prominent in pristane-vs. MO-treated mice, suggesting that pristane causes neutrophil apoptosis. Staining with an antibody against activated caspase-3 was not increased in monocytes from pristane-treated mice, suggesting that pristane may not induce apoptosis in these cells.
Plasma membrane asymmetry is maintained by flippases, such as ATP11C, and phospholipid scramblases, such as ANO6 (TMEM16F) and XKR8, which help retain PtdSer in the inner leaflet (Segawa and Nagata, 2015). In apoptotic cells, cleavage by caspases irreversibly inactivates ATP11C flippase and activates XKR8 scramblase, resulting in increased PtdSer exposure on the cell surface. However, in activated cells, increased intracellular Ca 2+ can reversibly activate TMEM16F and inactivate ATP11C, resulting in transiently increased concentrations of PtdSer in the outer leaflet without apoptosis (Segawa and Nagata, 2015;Doktorova et al., 2020). Loss of plasma membrane asymmetry plays a role in immune signaling, including the release of cytokine-loaded microvesicles from monocytes (MacKenzie et al., 2001). We conclude that pristane may selectively alter the activation state of monocytes, leading to a reversible loss of plasma membrane asymmetry. Increased CD138 expression may be marker for this activated state, which is associated with increased production of inflammatory cytokines ( Figure 7C), possibly via a mechanism similar to that reported for IL-1β (MacKenzie et al., 2001).
What is the role of CD138?
The function of CD138 (syndecan-1, Sdc1) in myeloid cells is uncertain. Although the protein is expressed by a subset of Ly6C − monocytes and Mϕ, Sdc1 is not among the genes differentially expressed in Ly6C hi vs. Ly6C − monocytes from non-pristane-treated-mice (Mildner et al., 2017). There are several plausible explanations. First, the antibody we used might cross-react with another target in myeloid cells. However, clone 281-2 is a standard antibody for flow cytometry of mouse CD138 that recognizes the extracellular domain of CD138 on plasma cells, epithelial cells, and early B cells (Jalkanen et al., 1985). Although transcriptional profiling of Ly6C − CD138 + monocytes was not done, flow-sorted CD138 + Mϕ express high levels of Sdc1 mRNA (Han et al., 2020) and others also have reported CD138 mRNA and protein expression in peritoneal Mϕ (Yeaman and Rapraeger, 1993). A more likely possibility is that Sdc1 is not normally expressed in monocytes, but is induced directly or indirectly by pristane.
CD138 is a heparan sulfate-containing membrane proteoglycan that binds extracellular matrix proteins and promotes integrin activation, wound healing, cell adhesion/migration, endocytosis, and fibrosis (Stepp et al., 2015). The extracellular domain binds type IV collagen and laminins (Stepp et al., 2015;San Antonio et al., 1994;Salmivirta et al., 1994;Jung et al., 2009), which are components of the basement membrane separating the alveolar capillary endothelium and the epithelium (Loscertales et al., 2016). In chronic inflammation, monocytes attach to the vascular endothelium via LFA-1, CX3CR1, and CD11b (Auffray et al., 2007;Gao et al., 2012), all of which were highly expressed by Ly6C − CD138 + monocytes ( Figure 5C and Figure 6C,D). They then migrate to the subendothelial space, and reversibly attach to basement membrane laminin via integrins (Kostidou et al., 2009a;Kostidou et al., 2009b). It will be of interest to investigate whether CD138 expression promotes monocyte migration and/or attachment to the basement membranes of damaged blood vessels.

Treml4 is transcriptionally regulated by Nr4a1
Nr4a1 (Nur77) is a member of the nuclear receptor 4A subfamily that regulates monocyte and Mϕ differentiation (McEvoy et al., 2017). It is highly expressed in Ly6C − monocytes, which are deficient in Nr4a1−/− mice (Hanna et al., 2011;Thomas et al., 2016). Our data suggest that Treml4 is regulated by Nr4a1. Treml4 mRNA and protein levels correlated with Nr4a1 expression in pristane-treated mice and in TLR ligand-or pristane-treated RAW264.7 cells (Figures 8 and 10). Conversely, Nr4a1 knockdown decreased Treml4 expression in RAW264.7 cells and Treml4 expression was lower in adherent Mϕ from Nr4a1−/− mice vs. controls ( Figure 10). Although Nr4a1 appears to transcriptionally regulate Treml4, additional factors are likely to be involved, since low levels of Treml4 were expressed in Ly6C − CD138 + and Ly6C − CD138 − monocytes and Mϕ from Nr4a1−/− mice (Figure 8). Relevant to the role of Ly6C − monocytes in monitoring endothelial damage, TremL4 binds late apoptotic and necrotic cells and sensitizes myeloid cells to TLR7 signaling by recruiting MyD88 to endosomes (Hemmi et al., 2009;Ramirez-Ortiz et al., 2015). Thus, high levels of TremL4 may promote inflammatory cytokine production by monocytes infiltrating the lung in mice with DAH. TremL4 also may play a role in lupus nephritis. Treml4−/− MRL/lpr mice have lower autoantibody and interferon-α production and improved survival vs. Treml4+/+ controls (Ramirez-Ortiz et al., 2015), suggesting that Treml4 plays a causal role in lupus nephritis by upregulating TLR7-driven interferon production. Since patrolling monocytes play a role in glomerular inflammation in murine lupus nephritis (Kuriakose et al., 2019), Treml4 expression may enhance the inflammatory response to injured glomerular endothelial cells.
Genetic polymorphisms that increase TREML4 mRNA expression in human peripheral blood cells are associated with the progression and extent of coronary atherosclerotic lesions (Duarte et al., 2019). However, a causal relationship has not been established and most evidence suggests that patrolling monocytes are protective in atherosclerosis (Hanna et al., 2012;Narasimhan et al., 2019). It is possible that upregulation of TLR7 signaling by Treml4 has a dual effect, on one hand promoting vascular integrity by enhancing the removal of damaged endothelial cells, and on the other enhancing vascular inflammation if the endothelial cell damage cannot be resolved.
In summary, DAH, a manifestation of pristane-induced lupus mediated by monocytes, was abolished in the absence of Ly6C hi inflammatory monocytes. However, Ly6C hi monocytes also increased in MO-treated mice without DAH, suggesting that Ly6C − monocytes play a secondary role. Our data suggest that patrolling monocytes from pristane-and MO-treated mice assume alternative, proinflammatory vs. proresolving phenotypes, respectively. Circulating Nr4a1-dependent Ly6C − CD138 − monocytes in MO-treated mice were associated with resistance to DAH, whereas Ly6C − CD138 + monocytes were associated with susceptibility. CD138 may be a marker for an activated subtype of patrolling monocytes, as also suggested by the increased annexin-V staining of monocytes from pristane-vs. MO-treated mice. In pristane-treated mice annexin-V staining also was higher in Ly6C hi monocytes, the precursor of Ly6C lo patrolling monocytes. Thus, pristane may alter the precursors of Ly6C lo patrolling monocytes, either in the BM or immediately upon egress of those cells from the marrow. Patrolling Ly6C lo CD138 + annexinV hi monocytes with high Treml4 expression may cause an exaggerated inflammatory response to damaged lung endothelial cells. In contrast, Ly6C − CD138 − annexinV lo monocytes with low Treml4 expression may be less inflammatory, facilitating the repair of lung vascular damage and protecting from DAH.  . There were 5-15 mice female mice age 8-12 weeks per group unless otherwise noted. Experiments were repeated at least twice. Peritoneal exudate cells (PECs) were collected by lavage 14 days later and heparinized blood was collected from the heart 3-14 days after pristane injection. DAH was assessed as described . This study followed the recommendations of the Animal Welfare Act and US Government Principles for the Utilization and Care of Vertebrate Animals and was approved by the UF IACUC.

Flow cytometry
Flow cytometry of peritoneal cells was performed using anti-mouse CD16/32 (Fc Block, BD Biosciences, San Jose, CA) before staining with primary antibody or isotype controls. Peripheral blood (100 µl) was incubated 30 min in the dark with monoclonal antibodies specific for surface markers. After surface staining, cells were fixed/permeabilized for 5 min (Fix-Perm buffer, eBioscience, San Diego, CA), washed and stained intracellularly for 20 min with anti-Nr4a1 monoclonal antibodies. The cells were washed and resuspended in PBS for flow cytometry. Monoclonal antibodies are listed in Table 2.
In other experiments, B6 mice were treated with either pristane or MO and 14 days later, blood was collected by cardiac puncture. Erythrocytes were lysed and leukocytes were cultured for 20 hr in low attachment tissue culture plates containing complete DMEM + 10% fetal bovine serum plus R848 (1 µg/ml) or vehicle. Cells were stained with FITC-conjugated anti-Ly6C, APC-conjugated anti-CD138, Bv-421-conjugated anti-CD11b, and APC-Cy7-conjugated anti-Ly6G antibodies followed by staining with PE-conjugated annexin-V (PE Annexin V Apoptosis Detection Kit, BioLegend), using the manufacturer's recommended buffer and protocol. RAW264.7 cell culture with TLR ligands RAW264.7 cells (murine Mϕ cell line, ATCC, Manassas, VA) were seeded in 24-well non-attachment plates (5 × 10 5 cells/well) and cultured for 24 hr in complete DMEM + 10% fetal bovine serum with or without LPS (1 µg/ml) or R848 (1 µg/ml). The cells were washed with PBS and analyzed by flow cytometry. Cells were surface stained for TremL4 and CD86 and intracellularly stained for Nr4a1.
For qPCR analysis, RAW264.7 cells were seeded in 6-well plates (3 × 10 5 cells/well) and cultured overnight in complete DMEM + 10% FBS. On the following day, the cells were stimulated with LPS (1 µg/ml) or R848 (1 µg/ml) or PBS for 1, 2, 3, 4, 5, or 6 hr. Total RNA was obtained using the QIAamp RNA blood Mini Kit (Qiagen, Germantown, MD), and cDNA was synthesized using the Superscript II First-Strand Synthesis kit (Invitrogen, Carlsbad, CA). SYBR Green qPCR analysis was performed using the CFX Connect Real-Time system (Bio-Rad, Hercules, CA). Gene expression was normalized to 18S RNA and the expression level was calculated using the 2 −ΔΔCt method.

RAW264.7 cell culture with pristane
Pristane or MO (1 ml) was added to PBS (9 ml) containing 100 mg/ml BSA in a 15 ml polypropylene tube and rotated for 48 hr at 4°C. The surface layer of unincorporated hydrocarbon oil was aspirated at the end of the incubation. The amount of pristane incorporated using this method was calculated as described  and adjusted to approximately 1 µg/ml. RAW264.7 cells were seeded in 24-well non-attachment plates (3 × 10 5 cells/well) and cultured for 24 hr in complete DMEM + 10% FBS with/without hydrocarbon oils at concentrations ranging from 12.5 to 200 ng/ml. Then cells were washed with PBS and analyzed by flow cytometry for surface TremL4 and intracellular Nr4a1.
Nr4a1 gene silencing RAW264.7 cells were seeded in 6-well plates (2 × 10 5 cells/well) and cultured overnight in antibioticfree complete DMEM + 10% FBS. On the following day the cells were transfected with Nr4a1 (Nur77) siRNA (sc-36110 from Santa Cruz Biotechnology) or control siRNA-A (sc-37007, Santa Cruz Biotechnology, Dallas RX). siRNA (40 pmol from a 10 µM stock in sterile water) was added to 100 µl of Opti-MEM medium and transfected using siRNA transfection reagent (sc-29528, Santa Cruz Biotechnology) following the manufacturer's instructions. Transfected cells were incubated at 37°C for 48 hr, and then stimulated with LPS (1 µg/ml) for 1, 3, or 6 hr. Total RNA was collected from the cells using the QIAamp RNA blood Mini Kit (Qiagen) and cDNA was synthesized using the Superscript II First-Strand Synthesis kit (Invitrogen). SYBR Green qPCR analysis was performed using the CFX Connect Real-Time system (Bio-Rad). Gene expression was normalized to 18S RNA and the expression level was calculated using the 2 −ΔΔCt method. Primer sequences were as above.

Peritoneal cell culture with TLR ligands
PECs were harvested from untreated wild-type B6 and Nr4a1−/− mice and allowed to adhere to plastic wells (6-well plates, 2 × 10 6 cells/well) for 1 hr in AIM V serum free medium (Thermo Fisher Scientific, Waltham, MA). Non-adherent cells were washed off with PBS and the adherent cells were cultured for 3 hr in complete DMEM + 10% FBS medium containing LPS (1 µg/ml in PBS) or PBS alone. Nr4a1 and Treml4 expression was quantified by qPCR as above.

Statistical analysis
Statistical analyses were performed using Prism 6.0 (GraphPad Software, San Diego, CA). Differences between groups were analyzed by two-sided unpaired Student's t-test unless otherwise indicated. Data were expressed as mean ± standard deviation. p < 0.05 was considered significant. Experiments were repeated at least twice.

Data availability
There are no datasets generated in this current manuscript. The results were put into figures (Figures  1-8).